The sensitivity of assays for measuring target molecules in a sample is generally constrained by the threshold of detection provided by the selected method. For example, typical detection systems for proteins employ an antibody specific for that protein immobilized on a solid support matrix. The matrix is typically a nitrocellulose filter, and the detection and quantification is based on antibody-protein binding such that the target protein is retained on the filter and then quantitated (usually by detection of a fluorescent or radioactive moiety present on the immobilized antibody, or incorporated in the target molecule). However, limitations in threshold detection levels exist in the art as it is presently known. For example, an antibody that binds specifically to a known protein can have a KD for that protein that is in the range of 10−9 molar concentration. If the protein is present at a concentration below the range of the KD, the antibody is not useful for such detection, since the antibody-antigen complex will not form to an appreciable extent and thus the target protein will not be retained on the solid support matrix. Further, formation of even one or a few antibody-antigen complexes cannot be detected in the presence of an excess of other materials. Techniques such as MALDI-TOF and similar analyses require expensive detection and analytical instrumentation which are not commonly available in a medical environment, and are complicated to operate on a high throughput basis.
Aptamers have been developed to bind specifically to target molecules for purposes of identifying the molecules for disease analysis. PCT application number WO 99-07724, by Nextar Pharmaceuticals, Inc., authored by Heilig and Gold, “Nucleic Acid Ligands for Blood-Brain and Cerebrospinal Fluid-Blood Barriers by Tissue SELEX,” published Feb. 18, 1999, discloses use of the SELEX system of obtaining a nucleic acid that has a sequence capable of binding a target protein with high affinity and specificity, in this case for components of cerebrospinal fluid and the blood-brain barrier. The nucleic acid having affinity for a target is known as an “aptamer”; aptamers have been developed for a variety of different types of target materials. See also, for example, PCT application number WO 95/07364, by Nexagen, Inc., authored by Gold et al., “Nucleic Acid Ligands and Improved Methods for Producing the Same,” published Mar. 16, 1995; and PCT application number WO 91/19813, by University of Colorado Foundation, authored by Gold and Tuerk, “Nucleic Acid Ligands,” published Dec. 26, 1991. The foregoing publications and the references cited therein are hereby incorporated herein by reference.
These methods typically use proteins and other molecules as the starting point, and then seek to identify, select, and enrich aptamers capable of binding the target molecules. Of particular interest to those using such methods is the identification of nucleic acid aptamers capable of binding molecules not previously known to bind nucleic acids.
Quantification of nucleic molecules has been achieved using Quantitative Polymerase Chain Reaction (QPCR), Quantitative Competitive Reverse Transcription-PCR [QC (RT)-PCR] or Real Time Detection 5′-Nuclease-PCR (RTDN-PCR; also known as TaqMan RT-PCR), is limited because of its focus on detection of nucleic acids. These techniques have been used to detect and measure concentrations of viable Cryptosporidium parvum oocytes present in environmental water concentrates, based on detection of DNA generated with RT-PCR from mRNA obtained from oocytes using oligo(T)25 magnetic beads (“Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocytes in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR,” C. Kauener and T. Stinear, Appl. Environ. Microbiol., 1998, 64(5): 1743–1756). However, detection is limited by the necessity of obtaining mRNA, an inherently unstable and short-lived species (t1/2 values in cells are typically less then 3 minutes for any given mRNA.)
It is also known in the art to correlate the concentration of a protein in a sample tissue to its mRNA expression, such as thrombopoietin in bone marrow cells, by use of TaqMan real-time quantitative RT-PCR (“Concentrations of Thrombopoietin in Bone Marrow in Normal Subjects and in Patients with Idiopathic Thrombocytopenic Purpura, Aplastic Anemia, and Essential Thrombocythemia Correlate With Its mRNA Expression of Bone Marrow Stromal Cells,” Y. Hirayama et al., Blood, 1998, 92(1): 46–52). This quantitative replicative method relies on the presence of a 5′-nuclease assay in the RT-PCR reactions, wherein a probe (the TaqMan probe) specific for an aptamer directed to the target protein, contains a fluorescent moiety such as 6-carboxyfluorescein (FAM) on the 5′-end, and a phosphate-capped quencher fluor moiety such as 6-carboxytetramethylfluorescein (TAMRA) on the 3′-end of the probe. As amplification of the mRNA for the target protein proceeds, the intensity of fluorescence of the FAM moiety increases as a function of time and mRNA concentration due to 5′-endonuclease cleavage of the probe, releasing more and more FAM moiety into the solution.
Again, this method is limited by the requirement for mRNA, and also is limited in that the concentration of thrombopoietin calculated in the sample is only a correlation to mRNA expression, it is not a measurement of thrombopoietin itself, or a measurement of a molecule which can be related directly to target protein concentration.
In other prior art, it is known to use either TaqMan RT-PCR coupled with an ABI Prism 7700 Sequence Detection System, or Competitive PCR for quantification of DNA from a bacterial pathogen in the sputum of patients during treatment (see “Comparison of the ABI 7700 System (TaqMan) and Competitive PCR for Quantification of IS6110 DNA in Sputum During Treatment of Tuberculosis,” Desjardin, L.e. et al., J. Clin. Microbiol. 1998, 36(7): 1964–1976). The two methods were found to be reproducible and accurate, and comparable in terms of detection limits. However, both methods were limited by poor correlation between quantity of M. tuberculosis DNA determined and the number of cultivable bacilli in the sputum specimen.
Quantitative replicative methods are thus focused on measurement of nucleic acids or its expression to determine protein concentration. Methods such as SELEX for generation, selection, and enrichment of nucleic acid aptamers are focused on the aptamer itself, e.g. finding aptamers which bind to unusual target molecules not previously known to bind nucleic acids, or finding aptamers which distinguish molecules of close chemical make-up, such as aptamers which can distinguish caffeine and theophyllin—molecules in the purine family which differ only in the presence or absence of a methyl group. In other methods, the nucleic acid aptamers are designed to prevent antibody interaction with its target receptor cell, in efforts to inhibit the allergic response (see for example, PCT application number WO 96/10576, by Nextar Pharmaceuticals, Inc. authored by Wiegand et al., “High Affinity Oligonucleotide Ligands to Immunoglobulin E (IgE)” published Apr. 11, 1996).